Team:Groningen/protocols/Transformation

From 2013.igem.org

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<h2><i>B. subtilis </i>transformation</h2>
<h2><i>B. subtilis </i>transformation</h2>
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<br><b>The Losick protocol is used</b>
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<H5>The Losick protocol is used</H5>
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<br>Day 0: Streak out the Bacillus strain of use and plate this on an LB agar plate o/n at 37°C.
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Day 0: Streak out the Bacillus strain of use and plate this on an LB agar plate o/n at 37°C.
<p>Transformation (D-Day):
<p>Transformation (D-Day):
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<p>
<p>
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<br/>Sub1: Competence medium (MC completed)
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<br/><H5>Sub1: Competence medium (MC completed)</H5>
<table border="1">
<table border="1">
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     <td>H2O?</td>
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     <td>H<sub>2</sub>O?</td>
     <td ALIGN=CENTER>1.8ml</td>
     <td ALIGN=CENTER>1.8ml</td>
     <td></td>
     <td></td>

Revision as of 18:52, 27 July 2013

B. subtilis transformation

The Losick protocol is used
Day 0: Streak out the Bacillus strain of use and plate this on an LB agar plate o/n at 37°C.

Transformation (D-Day):
1. Pick up a nice big colony and drop it in 2ml of completed MC (1x) (see sub1).
2. Grow at 37 °C for 5 hours (longer if the culture is not really turbid).
3. Mix 400µl of culture with DNA* in a fresh tube (i.e. 15ml tubes loosely closed – aeration)
4. Grow the cells at 37°C for an additional 2 hours .
5. Spread the complete 400µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight.

(*usually 1µl. Then 10µl of Quiagen plasmid miniprep or <1µl of chromosomal prep)


Sub1: Competence medium (MC completed)
compound amount treatment
H2O? 1.8ml
10x MC (sub2) 200µl filter sterilized
MgSO4 6.7µl autoclaved
Tryptophan 1% 10µl filter sterilized (stored in
aluminium foil

Sub2: MC 10x
for 100 ml 10 ml
K2H PO4 14,036g 1,4036g
KH2 PO4 5,239g 0,5239g
Glucose 20g 2g
Tri-Na Citrate 300mM(Sub3) 10ml 1ml
Ferric NH4 citrate(Sub4) 1ml 0,1ml
Casein Hydrolysate 1g 0,1g
Potassium Glutamate 2g 0,2g
Add 50ml H2O, Mix, add H2O till 100ml, Filter sterilize and freeze at -20 °C

Sub3: Tri-Na Citrate 300mM
Tri-Na Citrate 0,8823g
H2O 10ml
Wrap in aluminium

Sub4: Ferric NH4 citrate
Ferric NH4 citrate 0,22g
H2O 10ml
Wrap in aluminium